RNA-protein binding plays an important role in regulating protein activity by affecting localization and stability. While proteins are usually targeted via small molecules or other proteins, easy-to-design and synthesize small RNAs are a rather unexplored and promising venue. The problem is the lack of methods to generate RNA molecules that have the potential to bind to certain proteins. Here, we propose a method based on generative adversarial networks that learn to generate short RNA sequences with natural RNA-like properties such as GC content and free energy. Using an optimization technique, we fine-tune these sequences to have them bind to a target protein. We use RNA-protein binding prediction models from the literature to guide the model. We show that even if there is no available guide model trained specifically for the target protein, we can use models trained for similar proteins, such as proteins from the same family, to successfully generate a binding RNA molecule to the target protein. Using this approach, we generated piRNAs that are tailored to bind to SOX2 protein using models trained for its relative (SOX15, SOX14, and SOX7) and experimentally validated in vitro that the top-2 molecules we generated specifically bind to SOX2. We demonstrate that our generative model matched with the gradient-based optimization method is capable of generating piRNA sequences with high expected binding scores to the target protein. State-of-the-art RNA-Protein binding prediction models validate our results.

RNase Z from Deinococcus radiodurans couples tRNA processing with stress-responsive mRNA and sRNA turnover.

Tomato is an important vegetable crop that is rich in genetic variation, but published high-quality tomato reference genomes are limited. Small RNAs play ubiquitous roles in plants, but their biogenesis in tomatoes is not well studied. We assembled a high-quality chromosome-level reference genome for the tomato cultivar Ailsa Craig 57 (A57) and generated CRISPR mutants deficient in double-stranded RNA-binding protein 1 (SlDRB1) in this cultivar. Mutations in SlDRB1 led to severely abnormal development (e.g., loss of leaf polarity, pollen abortion, and infertility). High-throughput sRNA sequencing analysis revealed that SlDRB1, similar to its Arabidopsis ortholog Hyponastic Leaves 1 (HYL1), is required for the biogenesis of most miRNAs. Interestingly, SlDRB1 is also required for the accumulation of a large population of small RNAs derived from rRNAs and negatively regulates miR6026, which is dependent on DCL2 instead of DCL1. Comprehensive protein-protein interaction studies between SlDRB1 and SlDCL1 uncovered detailed interaction mechanisms involving subdomains of these two proteins. Their potential role in miRNA biogenesis is discussed.

Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary vascular remodeling, primarily driven by excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Although noncoding RNAs, such as circular RNAs, have been implicated in PH, the role of tRNA-derived small RNA remains poorly understood. Among them, i-tRF (internal tRNA-derived fragment)-Asp represents a novel tRNA-derived small RNA whose function in hypoxic PH is unknown. This study aims to investigate whether i-tRF-Asp contributes to PASMC proliferation and vascular remodeling in hypoxic PH. To elucidate the functional role of i-tRF-Asp, loss-of-function experiments were conducted in vitro using hypoxic mouse PASMCs, alongside in vivo studies using both SuHx (Sugen5416+hypoxia) induced PH models in both adult male C57BL/6 mice and Sprague-Dawley rats. These models employed antisense oligonucleotide inhibitors and adeno-associated virus-mediated silencing. The underlying mechanisms were further explored via RNA immunoprecipitation, liquid-liquid phase separation assays, cell proliferation assessment, Western blot, alternative splicing profiling, and molecular dynamics simulation. Inhibition of i-tRF-Asp ameliorated hypoxia-induced pulmonary vascular remodeling, primarily through suppressing excessive proliferation of mouse PASMCs. Further analysis indicated that i-tRF-Asp facilitates liquid-liquid phase separation of hnRNPU (heterogeneous nuclear ribonucleoprotein U). This process mediates selective recruitment of Tcf7l2 (transcription factor 7-like 2) pre-mRNA and promotes alternative splicing, resulting in a pro-proliferative Tcf7l2 transcript variant that drives mouse PASMC proliferation. Furthermore, we have identified that the endonuclease ERN1 (endoribonuclease IRE1) can upregulate the expression of i-tRF-Asp. The endoribonuclease ERN1 upregulates the expression of i-tRF-Asp, which in turn facilitates mouse PASMC proliferation and pulmonary vascular remodeling. This process is driven by hnRNPU liquid-liquid phase separation-dependent alternative splicing of the long Tcf7l2 transcript isoform.

Transfer RNA-derived small RNAs as potential biomarkers and therapeutic targets for autoimmune diseases.

MicroRNAs (miRNAs) play important roles in the pathogenesis of age-related macular degeneration (AMD), while whether polypoidal choroidal vasculopathy (PCV) represents a subtype of AMD remains controversial. However, the differential small non-coding RNA profiles in aqueous humor (AH) between neovascular AMD (nAMD) and PCV remain insufficiently characterized. Therefore, this study aimed to characterize miRNA and piRNA expression profiles in AH samples from nAMD and PCV patients and to explore the potential involvement of these small non-coding RNAs in angiogenesis-related pathways. AH samples were collected from nine cataract controls, eight treatment-naïve nAMD patients, and eight treatment-naïve PCV patients. Small RNA profiles in AH were analyzed using next-generation sequencing (NGS). Differential expression analysis was performed using DESeq2 with adjustment for age, sex, best-corrected visual acuity (BCVA), intraocular pressure (IOP), batch effects, and quality-control covariates. Target gene prediction, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were subsequently conducted. Selected miRNAs were partially validated by quantitative PCR (qPCR). To further evaluate their potential relevance to angiogenesis, expression levels of selected miRNAs were additionally examined in a laser-induced choroidal neovascularization (CNV) mouse model. A total of 35 differentially expressed miRNAs were identified between nAMD and PCV, including 28 upregulated and 7 downregulated miRNAs. Moreover, 27 and 47 uniquely expressed miRNAs were detected in nAMD and PCV, respectively. Four miRNAs exhibited opposite expression patterns between the two diseases. Functional enrichment analysis revealed significant involvement of Hippo, MAPK, and neurodegeneration-related signaling pathways. qPCR validation confirmed the differential expression of miR-150-5p and VEGF. In the laser-induced CNV mouse model, miR-150-5p showed expression changes consistent with the human AH sequencing results. Distinct miRNA and piRNA expression profiles were identified between nAMD and PCV, suggesting differential molecular mechanisms underlying the two diseases. These findings improve our understanding of AMD and PCV pathogenesis and may provide potential biomarkers for disease differentiation and angiogenesis-related research.

Overexpression of miR-455-3p enhances mitophagy, synaptic and mitochondrial proteins in Alzheimer's disease.

Comparative evaluation of four genetic markers for molecular detection of Giardia duodenalis in human fecal samples.

A roadmap for rapid detection of tuberculosis through microfluidic lab-on-chip strategies.

Neurodevelopmental disorders (NDDs) often remain unexplained due to limited assessment of non-coding genomic elements. Motivated by recent reports implicating RNU4-2, which encodes a spliceosomal small nuclear RNA (snRNA), we analyzed whole-genome sequencing data from 15 450 Korean individuals, including 2797 unrelated NDD probands. Rare pathogenic RNU4-2 variants were identified in 20 probands (0.72%), including 17 (85%) with a recurrent n.64_65insT variant. RNA secondary structure modeling and molecular dynamics simulations demonstrated that n.64_65insT disrupts the U4/U6 snRNA duplex and impairs exposure of the U6 ACAGAGA motif required for 5' splice-site recognition. Whole-blood RNA-seq from carriers revealed increased alternative 5' splice-site usage and dysregulation of immune, chromosomal, and DNA metabolic gene programs. Clinically, affected individuals presented with global developmental delay, microcephaly, seizures, failure to thrive, and dysmorphic features. These findings establish RNU4-2, particularly n.64_65insT, as a cause of early-onset NDD. We advocate for routine assessment of spliceosomal RNA genes in genomic diagnostics and reanalysis of unsolved cases to improve yield and guide counseling in rare neurodevelopmental syndromes.

Chronic consumption of (poly)phenol-rich foods exerts multigenomic modification of genes linked to cardiometabolic health in postmenopausal women.

Rhubarb attenuates intracerebral hemorrhage injury by upregulating Snord17 to inhibit PANoptosis.

Zoonotic Cryptosporidium and Giardia infections in pigeons (Columba spp.) at a wildlife hospital in France: Occurrence and molecular identification.

The role of miR-279b-5p regulating TcHex2 in hypoxic tolerance of Tribolium castaneum.

Transfer RNA-derived small RNAs (tsRNAs) represent a novel class of noncoding RNAs increasingly implicated in cardiovascular regulation. However, their roles in sepsis-induced cardiomyopathy (SICM) remain largely undefined. This study aimed to investigate the function and underlying mechanism of 5'tiRNA-32-LysCTT-11-a highly upregulated tsRNA in sepsis-induced cardiomyopathy-in modulating myocardial injury. A murine model of sepsis was established via cecal ligation and puncture, and myocardial injury was assessed by serum Creatine Kinase-MB Isoenzyme/lactate dehydrogenase levels, histology, and cardiac function via echocardiography. In vitro , H9C2 cardiomyocytes were exposed to conditioned media from lipopolysaccharide-stimulated macrophages. The expression of 5'tiRNA-32-LysCTT-11 was measured by quantitative reverse transcription polymerase chain reaction. Functional assays, including Cell Counting Kit-8, lactate dehydrogenase release, propidium iodide staining, 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine (JC-1), adenosine triphosphate, reactive oxygen species detection, and MitoTracker staining, were performed. Necroptosis was evaluated via mixed lineage kinase domain-like phosphorylation; mitochondria-associated endoplasmic reticulum membranes (MAMs) formation was assessed by dual-label immunofluorescence and phosphofurin acidic cluster sorting protein 2 expression. Bioinformatics analysis identified mitofusin 2 (Mfn2) as a putative target, validated by western blot, mRNA stability assay (actinomycin D), and rescue experiments. 5'tiRNA-32-LysCTT-11 was significantly upregulated insepsis-induced cardiomyopathy. In vivo , its overexpression improved cardiac function and reduced injury biomarkers. In vitro , 5'tiRNA-32-LysCTT-11 mimics preserved mitochondrial integrity, reduced reactive oxygen species and adenosine triphosphate depletion, suppressed mitochondria-associated endoplasmic reticulum membranes formation and necroptosis. Inhibitor transfection produced opposite effects. Mechanistically, 5'tiRNA-32-LysCTT-11 enhancedmitofusin 2 mRNA stability and protein expression. Silencing mitofusin 2 abrogated the protective effects, confirming its central role in the tsRNA's action. 5'tiRNA-32-LysCTT-11 exerts cardioprotective effects during sepsis by stabilizing Mfn2 mRNA, preserving mitochondrial function, limiting mitochondria-associated endoplasmic reticulum membranes formation, and suppressing necroptosis. These findings uncover a novel regulatory mechanism and suggest 5'tiRNA-32-LysCTT-11 as a promising therapeutic target SICM.

Engineering cis- and trans-acting RNA regulators for next-generation prokaryotic synthetic biology.

Small extracellular vesicles (EVs) from bovine milk and whey are emerging as biologically active carriers of microRNA (miRNA), yet their composition and variability among different dairy sources remain incompletely understood. We conducted an integrated characterisation of EV-associated miRNAs isolated from milk and whey collected from three dairy farms. EVs were purified and validated according to MISEV2023 criteria, and small RNA sequencing was performed on milk and whey samples collected on three different days at each farm (18 samples). After quality filtering and adaptor trimming, mapped reads were normalised to counts per million (CPM). To ensure robust detection, only miRNAs present in at least 67% of samples were retained, resulting in a high-confidence set of 329 miRNAs for downstream analyses. Milk-derived EVs showed a higher overall miRNA read count than whey-derived EVs (9.45±5.3 million vs. 5.03±2.5 million reads; p<0.10), with Farm Z displaying the greatest overall abundance. Stability assessment using Z-scored coefficients of variation demonstrated that miRNA reproducibility varied considerably between farms and collections, indicating that farm- and collection-specific factors, rather than matrix origin alone, were major contributors to expression variability. Differential expression analysis (DESeq2) identified 32 miRNAs significantly modulated between milk and whey EVs, with most (n=29) enriched in whey. Functional enrichment of predicted targets indicated involvement in immune and inflammation-related pathways, including NF-κB signalling, cytokine-receptor interaction and Ras signalling. Pairwise comparison of farms revealed only a small number of differentially expressed miRNAs in both matrices, and KEGG analyses did not identify significant pathways after multiple-testing correction. This study presents a comparative overview of bovine milk and whey EV-associated miRNAs, highlighting higher miRNA abundance in milk, significant farm-dependent variability, and enrichment of immune-related pathways that distinguish the two matrices.

Glioblastoma is a fatal primary malignant brain tumor, with an average survival of 15 months despite surgical resection, chemotherapy, and radiation therapy. Due to the concurrent deregulation of numerous genes in glioblastoma, molecular monotherapies have not improved clinical outcomes. Evidence suggests that targeting multiple deregulated molecules is essential for better therapies; however, this is limited by the lack of suitable drugs and increased toxicity of combination therapies. To address this, we hypothesized that miRNAs, small gene-regulatory RNAs that suppress mRNA, could simultaneously inhibit multiple deregulated genes in glioblastoma, and be used for more effective therapies. We identified regulatory miRNAs - those that target several deregulated genes in glioblastoma - using a combination of PAR-CLIP screening, TCGA data analyses and an algorithm to rank target importance and miRNA therapeutic potential. We selected two tumor suppressor miRNAs, miR-340 and miR-382, and one oncogenic miRNA, miR-17 and showed that they target critical glioblastoma pathways and alter cell growth, survival, invasion, and in vivo tumor growth. We developed and successfully applied a miRNA therapeutic delivery approach using Brain Penetrating Nanoparticles combined with MRI-guided focused ultrasound and microbubbles, to inhibit established tumor growth and to extend animal survival. This strategy offers a promising approach for translating miRNA-based therapies into clinical trials for glioblastoma and other cancers.

Despite excitotoxicity being a pivotal pathological mechanism in various retinal diseases, effective clinical interventions remain limited. Previous study has shown that adipose stem cell-derived extracellular vesicles (ADSC-EVs) can alleviate glutamate-induced retinal ganglion cells (RGCs) death by suppressing protein kinase C alpha (PKCA) pathway and increasing the expression of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors (AMPARs) subunit 2 (GluA2) on the cell membrane, but the mechanisms remain unexplored. To clarify the molecular processes involved in ADSC-EVs-mediated intracellular calcium balance, we isolated ADSC-EVs using ultrafiltration and ultracentrifugation, and characterized these vesicles by transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Small RNA sequencing was performed on glutamate-injured rat retinal precursor (R28) cells pre-treated with ADSC-EVs or PBS. Through bioinformatic analysis, we identified candidate microRNAs and predicted their potential target genes. The regulatory effects of microRNA were confirmed using propidium iodide staining, Fluo-4AM staining, western blotting, and immunofluorescence. Additionally, the RGCs counting and visual function tests were employed to evaluate the therapeutic efficacy of the microRNA in the glutamate-induced SD rat - animal model. Our results demonstrated that pre-treatment with ADSC-EVs led to a significant increase in the expression levels of miR-127-3p, miR-181b-1-3p, miR-199a-3p/5p, miR-23a-5p, miR-23b-5p, and miR-486 in R28 cells. Bioinformatic analyses suggest that miR-23a-5p may contribute to regulating the calcium overload by inhibiting the expression of phospholipase C delta 1 (PLCD1). Overexpression of miR-23a-5p or pre-treatment with ADSC-EVs modulated the expression of GluA2 on the cell membrane through inhibiting the PLCD1/PKCA/GluA2 axis, thereby reducing intracellular calcium levels and diminishing R28 cell death. In contrast, inhibition of miR-23a-5p expression partially reversed the regulatory effects of ADSC-EVs on calcium concentration and cell viability. Furthermore, our findings suggest that overexpression of miR-23a-5p in retina alleviated glutamate-induced RGCs death and visual function impairment, whereas suppression of miR-23a-5p exacerbated glutamate-induced RGCs death. ADSC-EVs delived miR-23a-5p mitigate glutamate-induced RGCs damage by inhibiting the PLCD1/PKCA/GluA2 axis. Targeting this miR-23a-5p-mediated axis may therefore represent a promising therapeutic approach for excitotoxic RGCs injury.

Extracellular vesicles and EV-enriched extracellular particles are increasingly recognized as potential mediators of intercellular communication. In coral reef ecosystems, dinoflagellate symbionts of the family Symbiodiniaceae play central roles in host metabolism, environmental acclimation, and stress responses; however, the molecular composition of extracellular particles released by cultured Symbiodiniaceae remains poorly understood. In this study, a host-isolated Symbiodiniaceae culture derived from the sea anemone Exaiptasia diaphana was established under host-free laboratory conditions for EV-enriched particle isolation and characterization. Physicochemical analysis of the recovered extracellular particle fraction showed an average diameter of 534.1 ± 63.9nm, a polydispersity index of 0.733 ± 0.08, and an average zeta potential of - 14.57 ± 1.55 mV, indicating a heterogeneous and negatively charged extracellular particle suspension. Small RNA analysis showed that the EV-associated RNA fraction contained multiple RNA biotypes, with rRNA- and tRNA-derived reads representing the dominant annotated components, whereas miRNA-like sequences accounted for only a minor fraction of the annotated small RNA pool. Comparison against an antisense Exaiptasia diaphana CDS dataset further identified retained EV-associated small RNAs showing sequence complementarity to host coding transcripts, including cytohesin-1 and EPG5. These matches are interpreted as candidate sequence-level observations rather than evidence of functional host regulation. In parallel, miRNA-oriented analysis identified candidate miRNA-like sequences after downstream filtering. Proteomic profiling by LC-MS/MS generated 4,086 accession-level matches. After removal of uninformative entries and consolidation of duplicate protein names, 834 non-redundant named proteins were retained and classified into nine functional groups, including proteins involved in metabolism and energy production, transport and membrane trafficking, photosynthesis and chloroplast function, and signal transduction and regulation. Representative annotations included 14-3-3-related proteins, Rab/ARF family proteins, Hsp70, Hsp90, and oxygen-evolving enhancer protein. These results provide an initial qualitative molecular characterization of EV-associated molecules released by a host-isolated Symbiodiniaceae culture and serve as a baseline for future studies of algal extracellular communication and cnidarian-dinoflagellate symbiosis.